A major goal of the human genome project is the generation of a two to five centimorgan linkage map of the entire genome. To this end, a first step is the establishment of an index map of highly polymorphic markers 10-15 centimorgans-apart. Currently, over 2000 markers have been identified, but only a relatively small fraction are highly informative when analyzed by conventional techniques (RFLP analysis). The aim of this proposal is to utilize powerful new techniques to identify highly informative polymorphisms in existing markers, especially in short PCR-amplified DNA fragments termed sequence tagged sites (STSs) and in gene sequences. We will utilize two approaches to identify polymorphisms within STSs, GC-clamped denaturing gradient gel electrophoresis (DGGE)and single stranded conformational gel electrophoresis (SSCG). In addition, we will utilize two variations of denaturing gradient gel electrophoresis to identify polymorphisms which lie in close proximity to STSs, thus increasing the probability of identifying highly informative polymorphisms in a given region. Furthermore, we will use GC-clamped DGGE and SSCG to identify sequence polymorphisms within specific Alu sequences. Initially our efforts will concentrate on chromosomes 1, 4 and 21 with the plan to expand this project to include regions of other chromosomes which show a paucity of RFLPS. A particular effort will be made to make all polymorphisms identified in this study easily usable by and accessible to other laboratories. In addition, our utilization of several approaches for identifying single base polymorphisms will allow us to evaluate the utility, efficacy, reproducibility and comprehensiveness of the various approaches. This should prove useful to other investigators seeking to identify additional polymorphisms working towards the goal of a more comprehensive linkage map. In the second year of this project we will make a concerted effort to analyze about 100 STSs solicited from other laboratories, concentrating on gaps in the genome lacking highly informative polymorphisms.